1. Technical Field
The present invention relates to assaying mean corpuscular volume (MCV) in blood samples; more particularly the invention relates to reagents for, as well as a method of use in, MCV assays using a particle analyzer.
2. Description of Related Art
Automated hematological analyzers are now widely used in the area of clinical examination. As devices for screening patients in medical diagnostic procedures, they are designed for rapid analysis of blood constituents. Such devices can perform the multiple assays of a complete blood count (CBC), including such items as red blood cell (RBC) count, white blood cell (WBC) count, leukocyte classification, hemoglobin concentration (Hb), hematocrit (Ht) and platelet (PLT) count.
The MCV is one among vital items obtained by calculating from these cytometric assay values. The MCV is obtained as the hematocrit divided by the RBC count, that is, the value Ht/RBC, the hematocrit being the percentage of erythrocytes (red blood cells) in a unit volume of whole blood. The MCV, then is the average volume of a single red blood cell, and accordingly it is given in fL, femtoliters, or 10.sup.-15 L.
The hematological analyzers noted above employ flow cytometers. In flow cytometers, a diluted blood sample is discharged into a flow cell at high speed through a very thin nozzle, and the discharging flow from the nozzle is surrounded by the cylindrical flow of a sheathing liquid (laminar flow). By narrowing the laminar flow, the sample flow can thus be focused, to the point at which the flow is essentially a cell-by-cell linear succession. The cells, whose passage in the sample flow is thus controlled by the laminar flow, are then detected optically, by irradiating the flow with a laser beam, as well as electrically, by measuring the resistance or conductivity.
To prepare a whole blood sample for flow-cytometric assay in a hematological analyzer as described above, the sample generally must be treated with an anti-coagulant agent such as an EDTA salt, and diluted with a physiologically isotonic solution.
As diluents for diluting whole blood, there are in general such solutions as physiological saline, Ringer's solution, Locke's solution, and Tyrode's solution. In performing the above-described assays in a hematological analyzer, the foregoing diluents can be used. Ordinarily, however, diluents optimized for individual analyzers are employed.
A basic problem with the current technology in preparing whole blood samples for MCV assay is that the actual MCV changes with post-blood drawing elapsed time. The MCV value that is necessary and important in clinical diagnosis is that which would be obtained immediately after the blood sample is drawn, that is, that value which corresponds to the MCV of the original blood.
In practice, the MCV value as part of a CBC is not measured until as much as 72 hours have passed following drawing of the blood sample. At that point, however, blood samples as such no longer yield suitable MCV assay values. After the blood is drawn, the RBCs in the sample swell, such that MCV values measured with the post-drawing elapse of time are progressively larger than the original RBC mean volume. The original MCV is the actual measurement diagnostically required.
Japanese Pat. Publ. 8-33388 (1996), Japanese Pat. Publ. 7-82010 (1995), and Japanese Laid Open Pat. 8-122327 (1996) disclose various aqueous solutions containing nonionic surfactants. Nevertheless, whichever of these solutions is used as a flow-cytometer sheathing liquid, and the nonionic surfactants are added for eliminating air bubbles within the flow chamber.
Furthermore, Japanese Pat. Publ. 1-33780 (1989) discloses a diluent into which a nonionic surfactant has been added to neutralize the effect on erythrocytes of antiseptic agents, ultimately with the object of restraining post-dilution change in the measured MCV due to the antiseptic agents. Nevertheless, Japanese Pat. Publ. 1-33780 does not disclose a method of restraining change in measured MCV due to the passage time after drawing blood samples.
It would be an important advance in the art if a means could be found to bring about consistency with blood sample original values in hematocrit and RBC count measurements made up to 72 hours after the blood is drawn.